Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the growth of myeloid progenitor cells, as well as enhancing the functional activity of circulating neutrophils, monocytes, and eosinophils. Evidence in vitro and in vivo suggests that GM-CSF is a potent granulopoietin which plays a critical role in host defense and inflammation; clinical trials employing GM-CSF in cancer therapy, bone marrow transplantation, and immunodeficiency disorders, are currently in progress. We have previously characterized numerous biological functions of GM-CSF on both immature progenitor cells and mature neutrophils and eosinophils. We have identified a low number of high affinity GM- CSF binding sites on all responsive target cells, and characterized the roles of Ca++, pH, and arachidonic acid release in neutrophil priming by GM-GSF. In this Continuation Application, we propose to extend our studies on the GM-CSF receptor to obtain purified preparations for biochemical characterization. We propose to continue studies on signal transduction processes, and to explore the function of GTP-binding proteins, protein kinases, and diacylglycerol in GM-CSF action. Several strategies are proposed to isolate cDNA clones encoding the GM-CSF receptor; we will perform site-directed mutagenesis to identify and characterize the ligand binding and intracellular domains of the molecule. We will also characterize the expression of the receptor in primary cells and tissues, as well as experimentally addressing the results of overexpression of native and modified receptor molecules. In addition to our studies on the GM-CSF receptor and its membrane signal transduction mechanisms, we are studying specific genes whose expression may be directly induced by GM-CSF, including early response genes, as well as cellular oncogenes known to be expressed in myeloid cells. We will characterize the pattern of gene expression in both dividing and terminally differentiated cells in response to GM-CSF. The ultimate goal of this work is to identify one or more """"""""representative"""""""" GM-CSF-responsive genes. We will isolate genomic clones, prepare recombinant constructs containing the 5' promoter region and, by means of deletion analysis and site- directed mutagenesis, identify nucleotide sequences responsive to GM-CSF treatment. The goal of these studies is to fully characterize the response of both normal and neoplastic myeloid cells to the granulopoietin GM- CSF. The results will provide an enhanced understanding of the physiologic role of GM-CSF in granulopoiesis; this knowledge is essential to design rational therapeutic strategies employing GM- CSF.
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